Process for the overexpression of dehydrogenases

ABSTRACT

A process for the overexpression of dehydrogenases, especially for the overexpression of Δ 1 -dehydrogenases, in particular for the overexpression of 3-keto steroid-Δ 1 -dehydrogenases, as well as for the bacteria, plasmids and DNA sequences that can be used for the overexpression, is described.

This invention relates to a process for the overexpression ofdehydrogenases, especially Δ¹-dehydrogenases, in particular 3-ketosteroid-Δ¹-dehydrogenases as well as the bacteria, plasmids and DNAsequences that are used for the overexpression.

The 3-keto steroid-Δ¹-dehydrogenase is an enzyme that fulfills animportant function in steroid metabolism. With the aid of this enzyme,the selective introduction of a double bond at 1-position in the steroidskeleton is made possible. This reaction is of great importance for thesynthesis of a wide variety of pharmaceutical active ingredients [e.g.,betamethasone, deflazacort, fluocortolone, hydroxy acid, prednisolone,etc.]. It would be desirable to make available large amounts of thisenzyme for a microbiological reaction.

For processes for microbial materials conversion, such as, e.g., steroidtransformations, wild strains of yeasts, fungi and bacteria aregenerally used [see, i.a., Kieslich, K. (1980), Steroid Conversions, In:Economic Microbiology—Microbial Enzymes and Transformation, Rose, A. H.(ed.), Academic Press, London, Vol. V, pp. 370-453; Kieslich, K. andSebek, O. K. (1980) Microbal Transformations of Steroids, In: AnnualReports on Fermentation Processes, Perlman, D. (ed.), Academic Press,New York, Vol. 3, pp. 275-304; Kieslich, K. (ed.) (1984)Biotransformation, Biotechnology, Vol. 6a, Rehm, H. J. and Reed, G.(eds.), Verlag Chemie, Weinheim]. In isolated cases, mutants that arealso derived from wild strains and that are obtained by standardmutagenesis and selection processes are used [see, i.a., U.S. Pat. No.3,102,080; Seidel, L. and Hörhold, C. (1992) J Basic Microbiol 32:49-55;EP 0322081 B1; U.S. Pat. No. 5,298,398]. Thus, e.g., in biotechnologicalprocesses for selective dehydrogenation, the endogenic catalyticactivity of different microorganisms, i.a., Arthrobacter simplex andBacillus sphaericus, is used [Sedlaczek (1988) Crit Rev Biotechnol.7:187-236; U.S. Pat. No. 2,837,464; U.S. Pat. No. 3,010,876; U.S. Pat.No. 3,102,080].

It is also known that Δ¹-dehydrogenase genes of Arthrobacter simplex[Choi, K. P. et al. (1995) J Biochem 117:1043-1049; Molnar, I. et al.(1995) Mol Microbiol 15:895-905], Comamonas testosteroni [Plesiat, P. etal. (1991) J Bacteriol 173:7219-7227] and Nocardia opaca [Drobnic, K. etal. (1993) Biochem Biophys Res Com 190:509-515; SUISS-PROT AC: Q04616]were cloned, sequenced and functionally characterized. Also, DNAsequences were published from Mycobacterium tuberculosis and Rhodococcusrhodochrous, and because of their similarity to the above-mentionedΔ¹-dehydrogenase genes, said sequences can be considered as presumabledehydrogenase genes [http://www.sanger.ac.uk/Projects/M_tuberculosis;GenBank AC: 007847].

Limitation of the known biotransformation processes lies in the factthat the latter are in general process optimizations that areconcentrated predominantly in the improvement of reaction conditions andprocess parameters, such as, e.g., type and composition of nutrients,execution of the process, substrate administration, etc. In particular,the processes for selective dehydrogenation have a number of drawbacks,such as, e.g., i) complete reaction of the educt only at very lowsubstrate concentrations [U.S. Pat. No. 3,102,080], ii) long operatingtimes, and iii) the formation of secondary zones—such as, e.g.,11α-hydroxyandrosta-1,4-diene-3,17-dione in the reaction ofhydrocortisone to form prednisolone, which must be separated byexpensive purification processes. These drawbacks result in the factthat the production process is very expensive.

It has now been found that by directed alteration of the microorganismsthat catalyze the materials conversion with molecular-biologicalmethods, better, more efficient and purposeful biotransformations ofsteroid molecules can be achieved. The biotransformation reactions areperformed with bacteria that contain plasmids for overexpression of3-keto steroid-Δ¹-dehydrogenase genes.

The bacteria that are used include in particular representatives of thegram-positive genus Bacillus, such as Bacillus subtilis, Bacillussphaericus, Bacillus lichen form is, Bacillus lentus and Bacillusmegaterium, but also gram-negative representatives, such as Escherichiacoli and Pseudomonas species.

By directed strain development with molecular-biological methods,microorganisms are designed that accelerate and simplify the synthesesof active ingredients, by i) the use of very high substrateconcentrations with ii) unaltered operating times being possible,without iii) disruptive secondary zones being developed.

In particular, selective dehydrogenation at 1-position of the steroidskeleton is described here, whereby 3-keto steroid-Δ¹-dehydrogenasegenes that are isolated from microorganisms are used.

According to the invention, a process for selective introduction of adouble bond into a steroid skeleton by overexpression of dehydrogenasesis now described, which is characterized in that

-   -   a) a dehydrogenase gene is isolated from a bacterium, cloned and        amplified,    -   b) promoter and terminator elements of the dehydrogenase gene or        other promoter and terminator elements are isolated from the        same or another bacterium, cloned and amplified,    -   c) expression plasmids are designed in which the dehydrogenase        gene from a), flanked by promoter and terminator sequences of        the dehydrogenase gene or by other promoter and terminator        elements from b), is contained,    -   d) bacteria are transformed with the expression plasmid that is        produced under c), and    -   e) the thus produced bacteria are cultivated, and the selective        dehydrogenation in the steroid skeleton is performed with these        cultures, whereby        -   i) a high substrate concentration at unaltered operating            times is used, and        -   ii) no disruptive secondary zones are produced.

This invention relates in particular to a process for selectiveintroduction of a double bond into a steroid skeleton by overexpressionof Δ¹-dehydrogenases, which is characterized in that

-   -   a) a Δ¹-dehydrogenase gene is isolated from a bacterium, cloned        and amplified,    -   b) promoter and terminator elements of the Δ¹-dehydrogenase gene        or other promoter and terminator elements are isolated from the        same or another bacterium, cloned and amplified,    -   c) expression plasmids are designed, in which the        Δ¹-dehydrogenase gene from a), flanked by promoter and        terminator sequences of the Δ¹-dehydrogenase gene of by other        promoter and terminator elements from b), is contained,    -   d) bacteria are transformed with the expression plasmid that is        produced under c), and    -   e) the thus produced bacteria are cultivated, and the selective        dehydrogenation in the steroid skeleton is performed with these        cultures, whereby        -   i) a high substrate concentration at unaltered operating            times is used, and        -   ii) no disruptive secondary zones are produced

This invention relates in particular to a process for selectiveintroduction of a double bond in a steroid skeleton by overexpression of3-keto steroid-Δ¹-dehydrogenases, which is characterized in that

-   -   a) the 3-keto steroid-Δ¹-dehydrogenase gene is isolated from a        bacterium, cloned and amplified,    -   b) promoter and terminator elements of the 3-keto        steroid-Δ¹-dehydrogenase gene or other promoter and terminator        elements are isolated from the same or another bacterium, cloned        and amplified,    -   c) expression plasmids are designed, in which the 3-keto        steroid-Δ¹-dehydrogenase gene from a), flanked by promoter and        terminator sequences of the 3-keto steroid-Δ¹-dehydrogenase gene        or by other promoter and terminator elements from b), is        contained,    -   d) bacteria are transformed with the expression plasmid that is        produced under c), and    -   e) the thus produced bacteria are cultivated, and the selective        dehydrogenation at 1-position in the steroid skeleton is        performed with these cultures, whereby        -   i) a high substrate concentration at unaltered operating            times is used, and        -   ii) no disruptive secondary zones are produced.

The bacteria that are mentioned in process steps a), b) and d) can beamong the gram-positive genus Bacillus, such as Bacillus spec., Bacillussubtilis, Bacillus sphaericus, Bacillus megaterium, Bacilluslicheniformis, Bacillus lentus as well as the gram-positiverepresentatives Arthrobacter simplex and Brevibacterium maris or thegram-negative representatives Escherichia coli and Pseudomonas species.

This invention relates in particular to the 3-ketosteroid-Δ¹-dehydrogenase gene from Arthrobacter simplex according toSeq. ID No. 1, the 3-keto steroid-Δ¹-dehydrogenase gene from Bacillussphaericus with promoter and terminator elements according to Seq. IDNo. 9 or Seq. ID No. 10, and the 3-keto steroid-Δ¹-dehydrogenase genefrom Brevibacterium maris according to Seq. ID No. 12 as well as thecorrespondingly expressed proteins, such as 3-ketosteroid-Δ¹-dehydrogenase from Bacillus sphaericus according to Seq. IDNo. 11, 3-keto steroid-Δ¹-dehydrogenase from Brevibacterium marisaccording to Seq. ID No. 13 and 3-keto steroid-Δ¹-dehydrogenase fromArthrobacter simplex according to Seq. ID. No. 14.

The above-mentioned DNA sequences can be introduced into host cells withsuitable plasmids. Suitable host cells or recipients are, e.g.,gram-positive bacteria of the genus Bacillus that can be used for theoverexpression of Δ¹-dehydrogenases with the purpose of dehydrogenatingsteroid molecules selectively in a biotransformation reaction. Inparticular, species such as Bacillus sphaericus and Bacillus subtilisare suitable for this purpose.

The bacteria are also subjects of this invention.

To introduce the inventive DNA sequences into the host cells, plasmidsare used that contain at least one of the above-mentioned DNA sequences.In the plasmids, the Δ¹-dehydrogenase genes are provided with suitablepromoters and terminators, which are necessary for overexpression inbacteria. Suitable promoter and terminators are, e.g., promoters andterminators of the 3-keto steroid-Δ¹-dehydrogenase gene of Bacillussphaericus according to Seq. ID No. 9, constitutive promoters such asp(veg) or promoters of bacteriophages Φ29 and SPO1, inducible promoterssuch as p(aprE) or p(sacB) from Bacillus subtilis, hybrid promoters suchas, e.g., a lacI-controlled SPO1-promoter, terminators of Escherichiacoli such as t(rrnB) or of Bacillus subtilis such as t(senS) or t(senN)[see, i.a., Doi, R. H. (1984) In: Biotechnology and Genetic EngineeringReviews, Vol. 2, Russell, G. E. (ed.), Intercept, Newcastle Upon Tyne,UK, pp. 121-153; Le Grice, S. F. J. et al. (1986) In: Bacillus MolecularGenetics and Biotechnology Applications, Ganesan, A. T. and Hoch, J. A.(eds.), Academic Press, New York, 433-445; Mountain, A. (1989) hi:Bacillus, Harwood, C. R. (ed.), Plenum Press, New York, pp. 73-114; LeGrice, S. F. J. (1990) Meth Enzymol 185:210-214; Wang and Doi (1992) In:Biology of Bacilli: Applications to Industry, Doi et al. (eds.),Massachusetts, Butterworth-Heinemann, pp. 143-188].

The plasmids are also subjects of this invention.

The plasmids can be used for transformation of bacteria that are capableof overexpression of Δ¹-dehydrogenases.

The invention also relates to DNA sequences with 3-ketosteroid-Δ¹-dehydrogenase activity, whose DNA sequences have a homologyof more than 80%, especially a homology of more than 90%, and preferablya homology of more than 95%.

The invention also relates to protein sequences with 3-ketosteroid-Δ¹-dehydrogenase activity that have a homology of at least 90%,especially a homology of at least 95%.

The invention also relates to promoters, especially the 3-ketosteroid-Δ¹-dehydrogenase promoter from Bacillus sphaericus with the DNAsequence Seq. ID. No. 9, as well as homologous promoters that have ahomology with Seq. ID No. 9 of more than 80%, preferably more than 90%,and especially preferably more than 95%.

The invention also relates to the Bacillus shaericus 3-ketosteroid-Δ¹-dehydrogenase oligonucleotides according to sequences Seq. IDNo. 15, Seq. ID No. 16, Seq. ID No. 17 and Seq. ID No. 18, and the parSoligonucleotides according to sequences Seq. ID No. 19 and Seq. ID No.20 and use thereof in processes for selective introduction of doublebonds into a steroid skeleton.

The DNA sequences and proteins according to the invention can be usedfor selective dehydrogenation of steroids. The DNA sequences and proteinsequences are also subjects of this invention.

Dehydrogenated steroids are, e.g., betamethasone, clobetasone,clocortolone,Δ¹-11β,17α-dihydroxy-6α,9α-difluoro-16α-methylprogesterone, deflazacort,dexamethasone, diflocortolone, fluocinolone acetonide, fluocortolone,hydroxy acid and prednisolone and derivatives of the above-mentionedcompounds.

Filings

The bacteria strains that are mentioned in the application can beordered from the respective filing sites, e.g., from DSM

Deutsche Sammlung von Mikroorganismen und Zellkulturen [GermanCollection of Microorganisms and Cell Cultures] GmbH, Mascheroder Weg1b, D-38124 Brunswick; ATCC

American Type Culture Collection, Rockville, Md., USA; NRRL

Northern Utilization Research and Development Division, Peoria, Ill.,USA; etc.

To better understand the invention that is based on this invention,first the methods that are used are described.

1. Restrictions

Restrictions of plasmid DNA and genomic DNA were performed in volumes of15 to 100 μl based on the amount of DNA that was used [1 to 20 μg]. Theenzyme concentration was 1 to 5 units of restriction enzyme per μg ofDNA. The reaction was performed in a buffer, incubated for one to threehours and subsequently analyzed on an agarose gel [Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.].

2. Agarose-Gel Electrophoresis

Gel electrophoreses were performed in Minigel-[BioRad],Midi-Widegel-[Biometra] and Maxigel devices [Biometra]. Depending on theseparating problem, agarose gels with 0.8% to 4% [w/v] agarose in0.5×TBE buffer were used. The electrophoresis was carried out with0.5×TBE as a running buffer. DNA fragments were stained with ethidiumbromide and made visible in a transilluminator [Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.]

3. Elution of DNA from an Agarose Gel

Preparative restriction preparations were separated in agarose gelaccording to size. The desired volumes were cut out with a scalpel. TheDNA fragment to be isolated was recovered with the aid of the “JetsorbKit” [Genomed] taking into consideration the instructions of themanufacturer and taken up in TE buffer.

4. Phosphorylation of Oligonucleotides

50 pmol of oligonucleotide was incubated in buffer recommended by themanufacturer in the presence of 0.1 mmol of ATP and 20 units of T4polynucleotide kinase for 45 minutes at 37° C. An enzyme inactivationwas carried out at 68° C. [Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.].

5. Ligation

For ligation, suitable amounts of dephosphorylated, linearizedvector-DNA and fragment-DNA were used in a molar ratio of 1:5. Thereaction was carried out in a volume of 10 μl with 1 unit ofT4-DNA-ligase in buffer recommended by the manufacturer at 16° C.overnight in a water bat [Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.].

6. Transformation of Escherichia coli

Competent E. coli cells were obtained by CaCl₂ treatment and stored at−80° C. In general, a 10 μl ligation stock was incubated with 200 μl ofcompetent cells. The transformation stocks were plated on LB agar withthe addition of antibiotic necessary in each case and incubated for 16hours at 37° C. Production of competent cells and a transformation werecarried out according to Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.

7. Transformation of Bacillus subtilis

The transformation of Bacillus subtilis was carried out according to thetwo-stage process described by Cutting, S. M. and Vander Horn, P. B.[In: Molecular Biological Methods for Bacillus (1990), Harwood, C. R.and Cutting, S. M. (eds.), John Wiley & Sons, Chichester].

8. Transformation of Bacillus sphaericus

Bacillus sphaericus was transformed by electroporation in a way similarto a process published by Taylor and Burke (1990) [FEMS Microbiol Lett66:125-1281. The cells were cultured overnight in MM2G medium [0.3%(w/v) meat extract, 0.8% (w/v) yeast extract, 1% (w/v) peptone, 0.2%(w/v) glucose, 0.7% (w/v) NaCl, 7.36 g/l of K₂HPO₄, 2.65 g/l of KH₂PO₄,5 m/l of 100% glycerol, pH 7], 1:20 was transferred into fresh MM2Gmedium, and it was cultivated for 90 minutes at 37° C. and 250 rpm. Thecells were pelletized, washed 3× with 10% glycerol and then taken up in750 μl of glycerol. 50 μl of cell suspension was mixed in anelectroporation cell with plasmid-DNA, incubated on ice, and placed inthe electroporation device [Biorad Gene Pulser™] [2.5 kV, 25 μF, 600Ω].The cells were incubated for regeneration for 90 minutes at 30° C. inMM2G medium and subsequently plated on TBAB agar/5 μg of neomycin[tryptose blood agar base (Difco)] and incubated for 24 hours at 30° C.

9. Plasmid Mini-Preparation from Escherichia coli

Mini-preparations were made according to the principle of alkaline celllysis [Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.].Individual colonies were cultured overnight in reagent glasses with 4 mlof LB medium and selection. 2 ml thereof was used for preparation.

10. Plasmid Mini-Preparation from Bacillus subtilis and Bacillussphaericus

The preparation of plasmids from Bacillus subtilis and Bacillussphaericus was carried out on columns of the Genomed Company [“JetstarKit Mini”] according to the protocol specified by the manufacturer. Toensure a complete cell lysis of the cells, the cell pellet that wastaken up in buffer E1 was mixed with 5 mg/ml of lysozyme, and the cellswere incubated for one hour at 37° C.

11. Plasmid Maxi-Preparation from Escherichia coli, Bacillus subtilisand Bacillus sphaericus

Plasmid maxi-preparation was made with the “Jetstar Kit Maxi” of theGenomed Company. The strains were cultivated overnight in 200 ml of LBmedium in the presence of an antibiotic. The preparation of the plasmidswas carried out according to the protocol specified by the manufacturer.To ensure a complete cell lysis of Bacillus subtilis and Bacillussphaericus, the cell pellets that were taken up in buffer E1 were mixedin addition with 5 mg/ml of lysozyme, and the cells were incubated forone hour at 37° C.

12. Preparation of Genomic DNA from Arthrobacter simplex, BacillusSpecies and Rhodococcus maris

200 ml of a densely-grown bacteria culture was pelletized and suspendedin 11 ml of solution I [50 mmol of Tris-HCl, pH 8; 50 mmol of EDTA; 1%(v/v) Triton x-100, 200 μg/ml of Rnase]. The suspension was mixed withlysozyme [5 mg/ml→A. simplex, B. sp./15 mg/ml→R. maris] and 500 μl ofproteinase K [20 mg/ml] and incubated for >30 minutes at 37° C. 4 ml ofsolution II [3 M guanidinium-hydrochloride, 20% (v/v) Tween] wassubsequently added thereto, and the stock was incubated for 30 minutesat 50° C. Undissolved particles were pelletized and discarded. Thechromosomal DNA that was dissolved in the lysate was purified by anionicexchange chromatography [“Jetstar Kit Maxi” of the Genomed Company, seethe protocol specified by the manufacturer].

13. Polymerase Chain Reaction

The reaction conditions for the PCR were optimized for each individualcase. In general, 0.1 to 0.5 μg of template-DNA, 10 mmol of dNTPs, 50pmol each of 5′- and 3′-primer as well as 2.5 units of Pwo-polymerase[Boehringer Mannheim] were combined in the buffer recommended by themanufacturer in 100 μl of total volume. Depending on the template-DNA,the stock was added up to 10% DMSO. The PCR was performed in a “BiometraTrio Thermoblock.” The temperature profile was newly modified for eachrequirement. The annealing temperature varied between 50° C. [lessstringent conditions] and 65° C. [See PCR 1: A Practical Approach,McPherson et al. (eds.), Oxford University Press (1991)]

14. Southern Analyses

In agarose gel, DNA that was separated according to size was transferredby the capillary-blot process [Sambrook et al. (1989) Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.] to positively-charged nylon membranes and linkedcovalently with the membrane by UV-irradiation.

Hybridizations were performed with digoxigenin-labeled probes. Thelabeling of the probes was carried out with the “DIG-High-Prime” or the“PCR DIG Probe Synthesis Kit” of Boehringer Mannheim according to theprotocol recommended by the manufacturer.

For hybridization, an SDS-phosphate buffer was used [7% SDS (w/v); 0.5 MNa phosphate, pH 7.0]. Depending on the requirements, stringent or lessstringent hybridization conditions were selected [Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.].

The detection of bonded DNA was carried out with a chemiluminescencereagent [CSPD®] of Boehringer Mannheim according to instructionsrecommended by the manufacturer.

14. Colony Hybridization

The transfer of colonies to Pall BIODYNE® A membranes [1.2 μm and 0.2 μmpore size] was performed according to the process recommended by themanufacturer.

The hybridization was carried out with digoxigenin-labeled probes in theabove-indicated SDS-phosphate buffer, and the detection was carried outwith a chemiluminescence reagent CSPD® of Boehringer Mannheim [“Pall BioSupport” application information SD1359G].

15. DNA-Sequence Analysis

DNA-sequence analyses were carried out with the GATC® 1500 system. Thesequence reactions were performed with the GATC®-BioCycle Sequencing Kitaccording to the protocol recommended by the manufacturer and analyzedon a 4% polyacrylamide-urea gel [GATC® 1500-system protocol]. Thedetection was carried out with CSPD® [GATC®-BioCycle Sequencing KitProtocol].

16. Hydrocortisone/Hydrocortisone-17-acetate→Prednisolone: Working-Upand Analysis

The culture broth was diluted with the 3× volume of methanol/1% aceticacid, ultrasound-treated and centrifuged off. The supernatant waschromatographed on an ODS-Hypersil column [250×4.6 mm] with anacetonitrile-water gradient at a flow rate of 1 ml/minute.

Sequence of eluants: hydrocortisone, prednisolone,11β-hydroxyandrosta-1,4-diene-3,17-dione, hydrocortisone-17-acetate,hydrocortisone-21-acetate, prednisolone-21-acetate.

17. 4-Androstene-3,17-dione→Androsta-1,4-diene-3,17-dione: Working-Upand Analysis

Isobutyl methyl ketone extracts of the culture broth were analyzed bygas chromatography:

Column 1: 50 m×0.25 mm, Chrompack WCOT CP5 CB, film thickness 0.4 μm

Column 2: 30 m×0.25 mm, hp 1701, film thickness 0.4 μm

Detector: FID

Carrier gas: hydrogen

Preliminary column pressure: 175 kPa

Sequence of the eluants: 4-androstene-3,17-dione,androsta-1,4-diene-3,17-dione

18. Fluocortolone A Acetate→Fluocortolone: Working-Up and Analysis

The culture broth was set at pH 4-6 with acetic acid and then extractedwith the 4× volume of isobutyl methyl ketone. The extract wasconcentrated by evaporation, taken up in chloroform and chromatographedon a Kromasil 100 column [250×4 mm] with an isocratic gradient ofchloroform:isooctane:1,4-dioxane:ethanol:water 1000:100:50:10:2 at aflow rate of 1.2 ml/minute.

Sequence of eluants: fluocortolone A acetate, fluocortolone A,fluocortolone

19.11β,17α-Dihydroxy-6α,9α-difluoro-16α-methylprogesterone→Δ¹-11β,17α-Dihydroxy-6α,9α-difluoro-16α-methylprogesterone:Working-Up and Analysis

The culture broth was diluted with the 3× volume of methanol/1% aceticacid, ultrasound-treated and centrifuged off. The supernatant waschromatographed on an ODS-Hypersil column [250×4.6 mm] with anacetonitrile-water gradient at a flow rate of 1 ml/minute.

Sequence of eluants:11α,17α-Dihydroxy-6α,9α-difluoro-16α-methylprogesterone,Δ¹-11β,17α-Dihydroxy-6α,9α-di-fluoro-16α-methylprogesterone

20.11β,21-Dihydroxy-2′-methyl-5′βH-pregn-4-eno[17,16-d]oxazole-3,20-dione→11β,21-Dihydroxy-2′-methyl-5′βH-pregna-1,4-dieno[17,16-d]oxazole-3,20-dione(Deflazacort Alcohol): Working-Up and Analysis

The culture broth was turraxed and then extracted with the 4× volume ofmethyl isobutyl ketone. The extract was evaporated to the dry state andtaken up in the same volume of chloroform. The sample was applied on aKromasil-100 column [250×4.6 mm] and chromatographed with diisopropylether:dichloroethane:1,4-dioxane:H₂O (250:150:75:4) at a flow rate of 2ml/minute. Sequence of the eluants:11β,21-dihydroxy-2′-methyl-5′βH-pregn-4-eno[17,16-d]oxazole-3,20-dione,11β,21-dihydroxy-2′-methyl-5′βH-pregna-1,4-dieno[17,16-d]oxazole-3,20-dione(deflazacort alcohol)

DESCRIPTION OF THE FIGURES

FIGS. 1 a/1 b shows the alignment of all known 3-ketosteroid-Δ¹-dehydrogenases

-   -   [CLUSTAL, W. Algorithmus, Thompson, J. D. et al. (1994) Nucleic        Acids Res 22:4673-4680].    -   In the figure:    -   Bm3os-delta1-DH means Brevibacterium maris 3-oxo        steroid-Δ¹-dehydrogenase    -   Rr3os-delta1-DH means Rhodococcus rhodochrous 3-oxo        steroid-Δ¹-dehydrogenase    -   As3os-delta1-DH means Arthrobacter simplex 3-oxo        steroid-Δ¹-dehydrogenase    -   Bs3os-delta1-DH means Bacillus sphaericus 3-oxo        steroid-Δ¹-dehydrogenase    -   Mt3os-delta1-DH means Mycobacterium tuberculosis 3-oxo        steroid-Δ¹-dehydrogenase    -   No3os-delta1-DH means Nocardia opaca 3-oxo        steroid-Δ¹-dehydrogenase    -   Ct3os-delta1-DH means Comamonas testosteroni 3-oxo        steroid-Δ¹-dehydrogenase    -   Number of perfect matches * 61        10.34%    -   Number of high similarity : 48        8.14%    -   Number of low similarity . 54        9.15%    -   Bm3os-delta1-DH [this work]; Rr3os-delta1-DH [GenBank AC:        AB007847];    -   As3os-delta1-DH [Molnar, I. et al. (1995) Mol Microbiol        15:895-905; GenBank AC: D37969]; Bs3os-delta1-DH [this work];        Mt3os-delta1-DH [cosmid Z82098, complement 16520 . . . 18211;        http://www.sanger.ac.uk/M_tuberculosis]; No3os-delta1-DH        [Drobnic, K. et al. (1993) Biochem Biophys Res Comm 190:509-515;        SUISS-PROT AC: Q04616]; Ct3os-delta1-DH [Plesiat P. et. (1991) J        Bacteriol 173:7219-7227; SUISS-PROT AC: Q06401].

FIG. 2 shows expression plasmid TS#196

FIG. 3 shows the reaction of EAF/MAF/F to form Pln [1 g/l]: cf. strain

-   -   AD#67 with Bacillus sphaericus ATCC 13805    -   In the figure:    -   EAF        Hydrocortisone-21-acetate    -   MAF        Hydrocortisone-17-acetate    -   F        Hydrocortisone    -   Pln        Prednisolone

FIG. 4 shows the reaction of EAF/MAF/F to form Pln [10 g/l]: cf. strain

-   -   AD#67 with Bacillus sphaericus ATCC 13805    -   In the figure:    -   EAF        Hydrocortisone-21-acetate    -   MAF        Hydrocortisone-17-acetate    -   F        Hydrocortisone    -   Pln        Prednisolone

FIG. 5 shows the reaction of AD to form ADD [1 g/l]: cf. strain AD#67with Bacillus sphaericus ATCC 13805

-   -   In the figure:    -   AD        4-Androstene-3,17-dione    -   ADD        Androsta-1,4-diene-3,17-dione

FIG. 6 shows the reaction of FCAA to form FC [1 g/l]: cf. strain AD#116with Bacillus sphaericus ATCC 13805

-   -   In the figure:    -   FCAA        Fluocortolone A acetate    -   FCA        Fluocortolone A    -   FC        Fluocortolone

FIG. 7 shows the reaction of DDFMP to form Δ¹-DDFMP [0.2 g/l]: cf.strain

-   -   AD#116 with Bacillus sphaericus ATCC 13805    -   In the figure:    -   DDFMP        11β,17α-Dihydroxy-6α,9α-difluoro-16α-methylprogesterone    -   Δ¹-DDFMP        Δ¹-11β,17α-Dihydroxy-6α,9α-difluoro-16α-methylprogesterone

FIG. 8 shows the conversion of EAF/MAF/F to Pln in 10 l of fermenter [20g/l]

-   -   Cf. strain AD#67/Bacillus sphaericus ATCC 13805    -   For the meaning of the abbreviations, see above.

FIG. 9 shows the reaction of11β,21-dihydroxy-2′-methyl-5′βH-pregn-4-eno[17,16-d]oxazole-3,20-dioneto form11β,21-dihydroxy-2′-methyl-5′βH-pregna-1,4-dieno[17,16-d]oxazole-3,20-dione(deflazacort alcohol) [1 g/L]: cf. strain

-   -   AO#205 with Bacillus sphaericus ATCC 13805

The cloning, isolation and construction examples below describe thebiological feasibility of the invention, without limiting the latter tothe examples.

EXAMPLE 1 Cloning of the 3-Keto Steroid-Δ¹-Dehydrogenase Genes fromVarious Species

1.1 From Arthrobacter simplex ATCC 6946

To isolate the 3-keto steroid-Δ¹-dehydrogenase gene from Arthrobactersimplex ATCC 6946, the open reader frame was amplified in a PCR reactionwith the primer pair 2026[5′ GGG GAT CCA TGG ACT GGG CAG AGG AGT ACG ACGTAC TGG TGG₁₄₃₅₋₁₄₆₈]and 2027 [5′ CGG AAT TCT CAT CGC GCG TCC TCG GTGCCC ATG TGC CGC ACG₂₉₈₂₋₂₉₄₉] from genomic DNA of Arthrobacter simplex.The amplified gene was cloned as an NcoI-EcoRI fragment in thecorresponding interfaces of vector pTrc99A [Pharmacia] or as aBamHI-EcoRI fragment in the corresponding interfaces of plasmid pSP72[Promega]. The gene sequence was verified with a GATC® 1500 Sequencer[GATC].

1.2 From Bacillus sphaericus ATCC 13805

To isolate the 3-keto steroid-Δ¹-dehydrogenase gene from Bacillussphaericus ATCC 13805, a homologous probe from genomic DNA of Bacillussphaericus was isolated with use of degenerated primers in a PCRreaction: under less stringent conditions, a 1463 bp fragment wasamplified with the primer pair 2048 [5′ GAA TRY GAT NTW NTW GTW GYW GGWWSW GG] and 2054 [5′ NAR NCC NCC YTT NGT NCC] and cloned in pCRScript™Amp SK(+) [Stratagene]. With the insert as a DNA probe, overlappinggenomic clones from a DNA library, which had been produced with the useof Zero Background™/Kan Cloning Kits [Invitrogen], were isolated. Thesequence of the Bacillus sphaericus 3-keto steroid-Δ¹-dehydrogenase genewas determined with a GATC® 1500 sequencer [GATC]. The protein sequencederived from the gene sequence is 34% identical to the sequence of the3-keto steroid-Δ¹-dehydrogenase from Comamonas testosteroni. Thesimilarity is 54%. A 34% identity and a 54% similarity exist in the3-keto steroid-Δ¹-dehydrogenase from Arthrobacter simplex.

1.3 From Brevibacterium maris ATCC 21111

To isolate the 3-keto steroid-Δ¹-dehydrogenase gene from Brevibacteriummaris ATCC 21111, first heterologous DNA probes were isolated from the3-keto steroid-Δ¹-dehydrogenase gene of Arthrobacter simplex andDIG-labeled: a 109 bp fragment [2066-2175] was amplified with the primerpair 2017 [GAC GCC GTA CTT CTG GCG GAG CTC GTC ATT GGC C₂₁₇₅₋₂₁₄₂] and2032 [CGA TCG TCG AGA CCG ACG G₂₀₆₆₋₂₀₈₄], a 190 bp fragment [1428-1618]was amplified with the primer pair 2016 [GAT CAC GAT GGA CTG GGC AGA GGAGTA CGA CG_(1428-1459]) and 2055 [GCA GCA CCG GGT TCG CGG GGA ACCAGG₁₆₁₈₋₁₅₉₂], and a 747 bp fragment [1428-2175] was amplified with theprimer pair 2016 and 2017. In Southern analyses, subsequent specificbinding of the above-mentioned probes to Brevibacterium maris DNA wasdetected. The conditions were used to identify clones with 3-ketosteroid-Δ¹-dehydrogenase gene sequences in a DNA library ofBrevibacterium maris, which had been produced with use of ZeroBackground™/Kan Cloning Kits [Invitrogen]. In this connection, twooverlapping clones were identified. The sequence of the Brevibacteriummaris 3-keto steroid-Δ¹-dehydrogenase gene was determined. The proteinsequence derived from the gene sequence is 28% identical to the sequenceof the 3-keto steroid-Δ¹-dehydrogenase from Comamonas testosteroni. Thesimilarity is 44%. A 72% identity and an 83% similarity exist in the3-keto steroid-Δ¹-dehydrogenase from Arthrobacter simplex.

A comparison of all known 3-keto steroid-Δ¹-dehydrogenases, includingnew sequences that are described here, yields—relative to the length ofthe consensus, an identity of only 10% and a similarity of only 18%[FIG. 1].

1.4 From Mycobacterium Species NRRL B-3683

For cloning the 3-keto steroid-Δ¹-dehydrogenase gene from Mycobacteriumspecies NRRLB-3683, first, analogously to the above, binding toMycobacterium sp. DNA was detected with the described DNA probes, andthe gene was then isolated from a genomic DNA library.

1.5 From Mycobacterium Species NRRL B-3805

For cloning the 3-keto steroid-Δ¹-dehydrogenase gene from Mycobacteriumspecies NRRLB-3805, first binding to Mycobacterium sp. DNA was detectedanalogously to the above with the described DNA probes, and the gene wasthen isolated from a genomic DNA library.

EXAMPLE 2 Isolating and Characterizing the Promoter and TerminatorSequences

As regulatory sequences for the overexpression of the 3-ketosteroid-Δ¹-dehydrogenase genes, promoter and terminator elements of the3-keto steroid-Δ¹-dehydrogenase gene from Bacillus sphaericus were used.Both elements were isolated and characterized in line with the cloningof the gene.

The promoter at position 84 bp or 61 bp above the startcodon containstwo hexanucleotides [TTGACT_(−84 to −79)/TATACT_(−61 to −56)], whichcorrespond, with a deviation in each case, to the consensus of bacterialpromoters [−10/−35 Box]. The distance from 17 nucleotides of the twoelements to one another corresponds exactly to the bacterial consensus[see Record, M. T. et al. (1996) In: Escherichia coli and Salmonella,Neidhardt, F. C. (ed.), 2^(nd) Edition, ASM Press, Washington D.C., Vol.1, pp. 792-821].

16 bp above the startcodon lies a ribosome-binding site that is typicalof Bacillus [AGGGAGG_(−16 to −10); Band, L. and Henner, D. J. (1984) DNA3: 17-21].

Promoter activity was detected for fragments from position −126 [SalI]to position −28 [ClaI] and from position −258 [PstI] to position −28[ClaI] in lacZ assays.

9 bp behind the stopcodon is a palindrome[AAGCCCTTCCT₁₆₉₈₋₁₇₀₈/AGGAAGGGCT₁₇₃₁₋₁₇₄₁], which acts as aρ-independent terminator [see Richardson, J. P. and Greenblatt, J.(1996) In: Escherichia coli and Salmonella, Neidhardt, F. C. (ed.),2^(nd) Edition, ASM Press, Washington D.C., Vol. 1, pp. 822-848].

In principle, other promoters and terminators can also be used [see,i.a., Doi, R. H. (1984) In: Biotechnology and Genetic EngineeringReviews, Vol. 2, Russell, G. E. (ed.), Intercept, Newcastle Upon Tyne,UK, pp. 121-153; Le Grice, S. F. J. et al. (1986) In: Bacillus MolecularGenetics and Biotechnology Applications, Ganesan, A. T. and Hoch, J. A.(eds.), Academic Press, New York, 433-445; Mountain, A. (1989) In:Bacillus, Harwood, C. R. (ed.), Plenum Press, New York, pp. 73-114; LeGrice, S. F. J. (11990)Meth Enzymol 185:210-214; Wang and Doi (1992) In:Biology of Bacilli: Applications to Industry, Doi et al. (eds.),Massachusetts, Butterworth-Heinemann, pp. 143-188].

EXAMPLE 3 Construction of Expression Plasmids

For the production of an expression plasmid, first a “shuttle” plasmidthat consists of pSP72 [Promega] and portions of pUB110 [McKenzie et al.(1986) Plasmid 15:93-103] was designed. To this end, pUB110 was cleavedwith EcoRI and PvuII, and the resulting 3.6 kb fragment was inserted inthe EcoRI and EcoRV interfaces of pSP72. The 3-ketosteroid-Δ¹-dehydrogenase gene of Bacillus sphaericus, flanked bypromoter and termination sequences [Position −126 (Sail) to Position1861 (ScaI)], was ligated as an XbaI-ScaI fragment in the XbaI and PvuIIinterfaces of the above-described “shuttle” vector [→TS#196, see FIG.2].

A second expression plasmid carries a modified Δ¹-dehydrogenase genepromoter p(Δ¹)_(mut): By PCR-mutagenesis, in each case a base wasexchanged in the −35 [TTGACT→TTGACA] and in the −10 Box [TATACT→TATAAT]to achieve an exact correspondence to the consensus of bacterialpromoters. For this purpose, the promoter was first amplified with themutagenesis primer 2089_(mut) [CCA TCG ATG AAT CTG GTC TTC CTA TTA AAAATT ATA GAA TTA AAC TAA TAT TCT GTC AAT TTT TCC_(−29 to −91)] and primer2090 [CAT GAC AAA ATT ATT TGA TTT AAT CAC_(−258 to −284)] and insertedas a PstI-ClaI fragment into the corresponding interfaces of pBluescriptII KS(+). The mutations were verified by sequence analysis. p(Δ¹)_(mut)was cut out as an XbaI-ClaI fragment and ligated in the correspondinginterfaces of TS#196. In this connection, the wt promoter was exchangedfor p(Δ¹)_(mut) [→TS#251].

In addition, two other plasmids carry a plasmid-stabilizing signal, parS[Lin, D. C. and Grossman, A. D. (1998) Cell 92:675-685]. The latter wascloned via two oligonucleotides that are complementary to one another,2091_(parS) [GAT CCT GTT CCA CGT GAA ACA G] and 2092_(parS) [GAT CCT GTTTCA CGT GGA ACA G], in the BamHI interface of TS#196 [→AD#82] and TS#251[→TS#255].

For expression in Escherichia coli DH5α[≡DSM 6897], the 3-ketosteroid-Δ¹-dehydrogenase gene of Bacillus sphaericus, flanked bypromoter and termination sequences, was cloned as a 2865 bp SalI-partialSau3A fragment [position −126 to position 2739] in the plasmid pZErO™-2that is cut with BamHI and XhoI and transformed into Escherichia coliDH5α [→plasmid MS#46 or; strain MS#46_(MS#461].)

EXAMPLE 4 Production of Recombinant Strains of the Genus Bacillus forthe Introduction of a Δ¹-Dehydrogenation on the Steroid

Expression plasmids TS#196, TS#251, AD#82 and TS#255 were transformedinto Bacillus subtilis DSM 402 [Deutsche Stammsammlung firMikroorganismen [German Strain Collection for Microorganisms],Brunswick] and Bacillus sphaericus ATCC 13805. Bacillus subtilis andBacillus sphaericus are gram-positive, apathogenic organisms. They aresimple to cultivate. In contrast to Bacillus sphaericus, Bacillussubtilis is well characterized in molecular-genetic terms. There are anumber of examples for the heterologous expression and secretion ofproteins for the production of recombinant gene products [Wang and Doi(1992) In: Biology of Bacilli: Applications to Industry, Doi et al.(eds.), Massachusetts, Butterworth-Heinemann, pp. 143-188]. Suitablepromoters and terminators are also described here.

With some of the recombinant strains, reactions of a mixture ofhydrocortisone [F], hydrocortisone-17-acetate [MAF] andhydrocortisone-21-acetate [EAF] to form prednisolone [Pln] wereperformed by way of example in a shaking flask. In addition to startingsubstances F, MAF and EAF as well as the desired product Pln, theformation of prednisolone-21-acetate [Pln-21-acetate] and theundesirable secondary zone 11β-hydroxyandrosta-1,4-diene-3,17-dione [11β-OH-ADD] was also tracked. To demonstrate the reaction potential of therecombinant strains, the process was performed at substrateconcentrations in which Bacillus sphaericus ATCC 13085 forms no morethan 20% Pln.

The strains AD#67_(TS#196), AD#94_(TS#251), AD#95_(TS#255),AD#96_(TS#255), AD#116_(TS#251), and AO#205_(TS#196) are produced fromBacillus sphaericus ATCC 13085 and in each case contain the indicatedexpression plasmid. Strains AD#89_(TS#196) and AD#90_(TS#196) areproduced from Bacillus subtilis DSM 402 and in each case contain theindicated expression plasmid.

The following reaction examples describe the microbiological feasibilityof the invention, without the latter being limited to the examples.

EXAMPLE 1 Reaction of EAF/MAF/F to Form Pln

Bacillus sphaericus ATCC 13805, AD#67_(TS#196), AD#94_(TS#251),AD#95_(TS#255), AD#96_(TS#255), AD#116 _(TS#251) , Bacillus subtilis DSM402, AD#89_(TS#196), AD#90_(TS#196) , Escherichia colt DH5α DSM 6897 andMS#46_(MS#46) were cultivated in LB medium [Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.] in the presence of 5 μg/ml of neomycin[Bacillus sphaericus derivatives], 50 μg/ml or 100 μg/ml of kanamycin[Escherichia coli or Bacillus subtilis derivatives] or without theaddition of an antibiotic [wt-strains] at 37° C. and 220 rpm. In thereaction of EAF/MAF/F to form Pln, the inoculation material 1:10 infresh LB medium was converted without the addition of antibiotic, andthe culture was shaken as above. In principle, any other medium in whichthe organism can grow can also be used. Substrate was added after 3hours. After 24 hours, the flasks were removed, and educts andproduct(s) were extracted and HPLC-analyzed [see Table 1; reactiondiagram, see below]. Bacillus subtilis DSM 402 and Escherichia coliDH5α, as expected, do not show any reaction, Bacillus sphaericus ATCC13085 forms less than 20% product after 24 hours, while all recombinantstrains of the genus Bacillus [AD#67_(TS#196), AD#94_(TS#255),AD#95_(TS#255), AD#96_(TS#255), AD#89_(TS#196) and AD#90_(TS#196)]produce more than 80% Pln in the same period. A degradation of substrateor product over 48 hours could not be observed.

All tests that are described below were performed by way of example withAD#67_(TS#196) or AD#116_(TS#251). As a standard, Bacillus sphaericusATCC 13085 was used. The tests show the reaction activity, increased bya multiple, of the above-mentioned recombinant strains with respect toΔ¹-dehydrogenations on the steroid molecule.

EXAMPLE 2 Kinetics of the Reaction of EAF/MAF/F to Form Pln [1 g/l]

First, a Δ¹-dehydrogenation in the example of a reaction of EAF/MAF/F toform Pln was performed analogously to the above at a substrateconcentration of 1 g/l in a shaking flask [LB medium, 37° C., 220 rpm].The addition of substrate was carried out after 3 hours. To be able totrack the course of the reaction, samples were taken after 4, 5, 6, 7,8, 9, 10, 11, 12 and 24 hours, and educts and products were extractedand HPLC-analyzed. While the strain ATCC 13805 requires 24 hours toconvert the substrate completely into Pln, strain AD#67 has alreadyformed the corresponding amount of Pln after <10 hours [FIG. 3; reactiondiagram, see below].

EXAMPLE 3 Kinetics of the Reaction of EAF/MAF/F to Form Pln [10 g/l]

The same test was performed at a substrate loading of 10 g/l. Thesubstrate was added after 3 hours, samples were taken after 6, 9, 12,24, 30 and 36 hours, and the steroids were extracted and analyzed. After6 hours, the ATCC 13085 culture has only 1% Pln, while the strain AD#67has already formed >15% product. After 12 hours, strain AD#67 hasalready converted more than 50% of the substrate into Pln; strain TCC13805, however, has converted only 5% [FIG. 4; reaction diagram, seebelow].

The high reaction activity of strain AD#67 is not limited to the processfor the production of prednisolone from EAF/MAF/F but rather applies ingeneral to the introduction of Δ¹ into a steroid molecule.

EXAMPLE 4 Conversion of 4-Androstene-3,17-dione [AD] intoAndrosta-1,2-diene-3,17-dione [ADD]

The conversion of AD into ADD by strain AD#67 or strain ATCC 13805 wasstudied analogously to the above in a shaking flask [LB medium, 37° C.,220 rpm]. The substrate was added after 3 hours, and samples were takenafter 4, 5, 6, 7, 9 and 10 hours. As in the conversion of MAF/F intoPln, the product formation is carried out considerably faster in thecase of fermentation with strain AD#67 than with use of strain ATCC13805. After 10 hours, Bacillus sphaericus ATCC 13805 has converted lessthan 30% of the substrate to ADD, while in strain AD#67 at this time,already more than 70% of product could be isolated [FIG. 5].

EXAMPLE 5 Reaction of Fluocortolone A Acetate [FCAA] to FormFluocortolone [FC]

Fluorinated steroids are also dehydrogenated by recombinant strainsconsiderably more efficiently in 1-position than was heretofore possiblewith the available bio-catalysts. This shows the conversion of FCAA toFC analogously to the above in a shaking flask by AD#116 in comparisonto Bacillus sphaericus ATCC 13805 [FIG. 6; reaction diagram, see below].

EXAMPLE 6 Reaction of11β,17α-Dihydroxy-6α,9α-difluoro-16α-methylprogesterone [DDFMP] to FormΔ¹-11β,17α-Dihydroxy-6α,9α-difluoro-16α-methylprogesterone [Δ¹DDFMP]

The conversion of DDFMP to Δ¹DDFMP analogously to the above-mentionedexamples is also carried out considerably more efficiently with AD#116than with Bacillus sphaericus ATCC 13805 [FIG. 7, reaction diagram, seebelow].

EXAMPLE 7 Conversion of EAF/MAF/F to Form Pln in a 10 l Fermenter [20g/l] Cf. Strain A)#67/Bacillus sphaericus ATCC 13805

The Δ¹-dehydrogenation capacity of strain AD#67 was tested in comparisonto Bacillus sphaericus ATCC 13805 in the example of EAF/MAF/F→Pln in 10l of fermenter. The reaction was performed in a 20× higher substrateloading. The culture of the inoculation material was carried out in afirst step overnight at 37° C. and 220 rpm in LB medium in the presenceof 5 μg/ml of neomycin [AD#67] or without the addition of an antibiotic[ATCC 13805]. Subsequently, the overnight culture 1:100 was convertedinto a 1000 ml intermediate culture and shaken for 9 hours at 37° C. and220 rpm to an optical density of 2.4. The fermentation was carried outin LB medium without the addition of an antibiotic. In principle,however, any other medium in which the organism can grow can be used.After 3 hours, the substrate was added continuously for 30 hours. The pHwas kept at 8. In the course of the fermentation, samples were taken andtested for the content of product and educt. The fermentation profileshows that Bacillus sphaericus ATCC 13805 cannot surmount substrateconcentrations of this order of magnitude: the reaction stops when morethan 80% substrate remains. The conversion capacity of strain AD#67,however, is considerable: Shortly after the substrate application phasehas ended, the reaction is almost fully [>98%] completed [FIG. 8]. Theconversion activity of strain AD#67 is approximately 0.6 g/l per hour.Strain ATCC 13805 shows, however, an activity of 0.1 g/l per hour. Inany case, disruptive secondary zones such as, e.g., 11-β-OH-ADD, wereobserved in traces. The crystal yield of Pln was approximately over 80%of theory and corresponds to the value that is achieved in conventionalprocesses [reaction diagram, see below].

EXAMPLE 8 Reaction of11β,21-Dihydroxy-2′-methyl-5′βH-pregn-4-eno[17,16-d]oxazole-3,20-dioneto form11β,21-dihydroxy-2′-methyl-5′βH-pregna-1,4-dieno[17,16-d]oxazole-3,20-dione(deflazacort alcohol)

The conversion of 1 g/l of11β,21-dihydroxy-2′-methyl-5′βH-pregn-4-eno[17,16-d]oxazole-3,20-dioneto deflazacort alcohol in a shaking flask analogously to theabove-mentioned examples is carried out significantly more efficientlywith AO#205 than with Bacillus sphaericus ATCC 13805 [FIG. 9; reactiondiagram, see below]. Unlike in the above, a medium that consists of 12g/l of 67% yeast extract, 27 g/l of corn steep liquor and 9.2 g/l ofNaCl was used.

TABLE 1 EAF/MAF Pln 11β-OH-ADD Substrate Strain F [mg/l] [mg/l] [mg/l][mg/l] Loading Bacillus sphaericus ATCC 13805^(a)) 7720 39 1730 <10 9g/l AD#67_(TS#196) ^(a)) 1570 22 7790 <10 9 g/l AD#94_(TS#251) ^(a))1650 30 7480 13 9 g/l AD#95_(TS#255) ^(a)) 1460 18 7500 13 9 g/lAD#96_(TS#255) ^(a)) 1530 19 7130 13 9 g/l AD#116_(TS#251) 2150n.d.^(b)) 9330 <10 12 g/l  Bacillus subtilis DSM 402^(a)) 9030 510 <1<10 9 g/l AD#89_(TS#196) 1820 500 8280 14 10 g/l  AD#90_(TS#196) 1680580 8120 <10 10 g/l  Escherichia coli DH5α 11110 1020 <1 n.d.^(b)) 12g/l  MS#46_(MS#46) 9510 1080 1910 n.d.^(b)) 12 g/l  ^(a))Doubledetermination ^(b))Not determined

1.-39. (canceled)
 40. A process for the selective introduction of adouble bond into ring A of a steroid skeleton by overexpression of3-keto steroid-Δ¹-dehydrogenases, comprising a) isolating aΔ¹-dehydrogenase gene from Arthrobacter simplex comprising Seq. ID No.1, a sequence having at least 90% homology to Seq. ID No. 1, Seq. ID No.14 or a sequence having at least 90% homology to Seq. ID No. 14, andcloning and amplifying said gene, b) isolating a promoter and aterminator element of the 3-keto steroid-Δ¹-dehydrogenase gene oranother promoter and terminator element from the same or anotherbacterium, and cloning and amplifying said elements, c) constructing anexpression plasmid comprising the 3-keto steroid-Δ¹-dehydrogenase genefrom a), flanked by promoter and terminator sequences of the 3-ketosteroid-Δ¹-dehydrogenase gene or by another promoter and terminatorelement from b), d) transforming host bacteria with the expressionplasmid that is produced under c), and e) cultivating the thus producedbacteria to dehydrogenate at the 1-position in the steroid skeleton,wherein i) a high substrate concentration at unaltered operating timesis used, and ii) no disruptive secondary zones are produced.
 41. Aprocess for selective introduction of a double bond into ring A of asteroid skeleton by overexpression of 3-keto steroid-Δ¹-dehydrogenases,wherein a) isolating an Δ¹-dehydrogenase gene from Brevibacterium mariscomprising Seq. ID No. 12, a sequence having at least 90% homology toSeq. ID No. 12, Seq. ID No. 13 or a sequence having at least 90%homology to Seq. ID No. 13, cloning and amplifying said gene, b)isolating a promoter and a terminator element of the 3-ketosteroid-Δ¹-dehydrogenase gene or isolating another promoter andterminator element from the same or another bacterium, cloning andamplifying said element, c) constructing an expression plasmid in whichthe 3-keto steroid-Δ¹-dehydrogenase gene from a) is flanked by apromoter and a terminator sequence of the 3-ketosteroid-Δ¹-dehydrogenase gene or by another promoter and terminatorelement from b), d) transforming a host bacteria with the expressionplasmid that is produced under c), and i) cultivating the thus producedbacteria, and selectively dehydrogenating the 1-position in the steroidskeleton, wherein ii) a high substrate concentration at unalteredoperating times is used, and no disruptive secondary zones are produced.42. A process according to claim 40, wherein said promoter comprisesSeq. ID No. 9 or a sequence having at least 90% homology to Seq. ID No.9.
 43. A process according to claim 41, wherein said promoter comprisesSeq. ID No. 9 or a sequence having at least 90% homology to Seq. ID No.9.
 44. A process according to claim 40, wherein said dehydrogenatedsteroid is betamethasone, clobetasone, clocortolone,Δ¹-11β,17α-dihydroxy-6α,9α-difluoro-16α-methylprogesterone, deflazacort,dexamethasone, diflocortolone, fluocinolone acetonide, fluocortolone,hydroxy acid or prednisolone and/or derivatives thereof.
 45. A processaccording to claim 41, wherein said dehydrogenated steroid isbetamethasone, clobetasone, clocortolone,Δ¹-11β,17α-dihydroxy-6α,9α-difluoro-16α-methylprogesterone, deflazacort,dexamethasone, diflocortolone, fluocinolone acetonide, fluocortolone,hydroxy acid or prednisolone and/or derivatives thereof.
 46. A processaccording to claim 40, wherein said steroid skeleton is hydrocortisone(F), hydrocortisone-17-acetate (MAF), hydrocortisone-21-acetate (EAF),4-androstene-3-17-dione (AD), fluocortolone A acetate (FCAA) or11β,17α-Dihydroxy-6α,9α-difluoro-16α-methylprogesterone (DDFMP).
 47. Aprocess according to claim 41, wherein said steroid skeleton ishydrocortisone (F), hydrocortisone-17-acetate (MAF),hydrocortisone-21-acetate (EAF), 4-androstene-3-17-dione (AD),fluocortolone A acetate (FCAA) or11β,17α-Dihydroxy-6α,9α-difluoro-16α-methylprogesterone (DDFMP).
 48. Aprocess according to claim 42, wherein said steroid skeleton ishydrocortisone (F), hydrocortisone-17-acetate (MAF),hydrocortisone-21-acetate (EAF), 4-androstene-3-17-dione (AD),fluocortolone A acetate (FCAA) or11β,17α-Dihydroxy-6α,9α-difluoro-16α-methylprogesterone (DDFMP).
 49. Aprocess according to claim 40, wherein said promoter or terminator is aconstitutive promoter that is p(veg), a promoter of bacteriophages Φ29or SPO1, an inducible promoter that is p(aprE) or p(sacB) from Bacillussubtilis, a hybrid promoter that is a lacI-controlled SPO1-promoter, aterminator of Escherichia coli that is t(rrnB) or of Bacillus subtilisthat is t(senS) or t(senN).
 50. A process according to claim 41, whereinsaid promoter or terminator is a constitutive promoter that is p(veg), apromoter of bacteriophages Φ29 or SPO1, an inducible promoter that isp(aprE) or p(sacB) from Bacillus subtilis, a hybrid promoter that is alacI-controlled SPO1-promoter, a terminator of Escherichia coli that ist(rrnB) or of Bacillus subtilis that is t(senS) or t(senN).
 51. Aprocess of claim 40, wherein said terminator is the terminator of the3-Keto steroid-Δ¹-dehydrogenase gene from Bacillus sphaericus comprisingSEQ ID NO:
 10. 52. A process of claim 41, wherein said terminator is theterminator of the 3-Keto steroid-Δ¹-dehydrogenase gene from Bacillussphaericus comprising SEQ ID NO:
 10. 53. A process of claim 40, whereinsaid host cell is B. sphaericus, B. subtilis or E. coli.
 54. A processof claim 41, wherein said host cell is B. sphaericus, B. subtilis or E.coli.